Introduction
Materials
Methods
Comments
Setting up the amplification reactions
Optimization
Choice of DNA polymerase
Applications
Introduction
Amplification-based nucleic acid scanning techniques driven by synthetic oligodeoxynucleotide primers of arbitrary sequence produce characteristic fingerprints capable of detecting sequence polymorphisms in anonymous nucleic acid templates (reviewed in Caetano-Anollés 1996,1998).The amplification of genomic DNA using at least one short primer usually results in multiple amplification products representing amplicons more or less randomly distributed throughout a genome (Livak et al. 1992, Bassam et al. 1995; McClelland et al. 1996). This observation led to the inception of three major techniques, randomly amplified polymorphic DNA (RAPD) (Williams et al. 1990), arbitrarily primed PCR (AP-PCR) (Welsh and McClelland 1990), and DNA amplification fingerprinting (DAF) (Caetano-Anolles et al. 1991). These methods became very popular because of their simplicity and wide applicability. A wide range of organisms have been studied and research reported in thousands of publications. Of these techniques, RAPD has been the most widely used. Its protocol is simple, with amplification products separated by agarose gel electrophoresis and visualized by ethidium bromide staining (Williams et al. 1993). Return
Materials
1. Equipment: thermocycler, agarose gel electrophoresis apparatus, power supply (with outputs of at least 200 V and 150 mA), ultraviolet transilluminator (light box), laboratory set-up for taking photographs of ethidium bromide-stained agarose gels or digital image capture system (e.g., CCD camera, computer and image analysis software), and spectrophotometer or fluorometer for determining DNA concentration
2. Water: Sterile, de-ionized or distilled water should be used for preparing all reagents and pre-mixes.
3. Reaction buffers: PCR buffer II (10¥: 100 mM Tris-HCl, 500 mM KCl, pH 8.3) and Stoffel buffer (10¥: 100 mM Tris-HCl, 100 mM KCl, pH 8.3), and standard PCR buffer (10¥: 100 mM Tris-HCl, 500 mM KCl, 15 mM MgCl2, 0.01% [w/v] gelatin, pH 8.3). When using standard PCR buffer adjust Mg+2 concentration appropriately. Store buffers at -20°C.
4. Deoxynucleoside triphosphates: 2 mM each of dGTP, dATP, dTTP, dCTP. Ready-made solutions of dNTPs are available from Pharmacia, Perkin-Elmer and many other suppliers. Aliquot into plastic test tubes and store at -20°C.
5. Primers: Decamer primers are available in sets from several suppliers (e.g. Operon, Genosys). Dilute in sterile distilled water to produce a 1-4 µM stock and store at -20°C.
6. Magnesium chloride: 25 mM solution prepared by dilution of a 1 M stock. Use sterile distilled water, aliquot and store at -20°C.
7. Taq DNA polymerase: AmpliTaq® (5 U/µl; Perkin-Elmer), Stoffel fragment of Taq DNA polymerase (10 U/µl; Perkin Elmer), or other high quality Taq polymerase
8. Genomic DNA: 5-25 ng/µl stocks. DNA of sufficient quality can be obtained by using the CTAB procedure (Murray and Thompson 1980) or established plant DNA isolation procedures (e.g., Chen and Dellaporta 1994, Dellaporta et al. 1985).
9. Reagents for agarose gel electrophoresis: agarose (e.g., SeaKem GTG from FMC Bioproducts, or similar), TBE buffer (1 M Tris-HCl, 0.9 M boric acid, 0.01 M EDTA pH 8.3), ethidium bromide DNA stain, . YO-PRO-1 iodide (1 mM in DMSO; Molecular Probes), and electrophoretic size standards (e.g. bacteriophage l DNA HindIII digest or bacteriophage fX174 HaeIII digest).
If DNA isolation problems arise, phenol extraction followed by ethanol precipitation is frequently helpful.
Contamination with DNA seen with some brands of Taq polymerase may cause appearance of bands in the "no DNA" controls. Return
Methods
1. Assemble RAPD reactions as follows: 5 µl DNA stock, 2.5 µl Stoffel buffer or PCR Buffer II, 2.5 µl or 1.7 µl magnesium chloride stock for Stoffel or Taq holoenzyme respectively, 2.5 µl of 4 µM primer stock, 1.25 µl of 2 mM dNTPs stock, 0.1-0.2 µl of either Stoffel fragment or Taq holoenzyme, and sterile distilled water to a total reaction volume of 25 µl. Wear gloves throughout the RAPD reaction preparation procedure. PCR buffer, dNTPs, MgCl2 solution and primer solutions are thawed from frozen stocks, mixed by vortexing and placed on ice. DNA, if stored frozen (see below) should also be thawed out and mixed gently. Assembled reactions are sealed, vortexed, centrifuged and placed in the thermocycler for DNA amplification. Add oil if recommended by the thermocycler manufacturer.
2. Amplify DNA in thermocycler. Cycling conditions may be modified depending on the thermocycler used. With average speed thermocyclers (e.g., Perkin-Elmer model 480) use 40 to 45 cycles of 1 min at 94°C, 1 min at 36°C, 2 min at 72°C, followed by 1 cycle of 7 min at 72°C and a 4°C incubation. With faster thermocyclers (e.g., Perkin-Elmer model 9600) a shorter protocol can be used: 40-45 cycles of 15 sec at 94°C, 30 sec at 36°C and 1 min at 72°C. An initial denaturation step of 3 min at 94°C and/or a final 7 min extension at 72°C can be added to the amplification protocol, depending on the templates used.
3. Prepare for agarose gel electrophoresis. Normally, use agarose gel concentrations of 1.4%, or adjust agarose concentration appropriately when analyzing the lower molecular weight amplification products obtained using Stoffel fragment. Higher concentrations can also be used routinely (up to 2.5% agarose or up to 3-4% of NuSieve® agarose, FMC Bioproducts). Include 0.5 µg/mL ethidium bromide in both gel and 1¥ TBE electrophoresis running buffer. Alternatively, stain the gel with ethidium bromide after electrophoresis.
4. Prepare and load samples for agarose gel electrophoresis. After the cycling is finished, add an appropriate volume of a gel loading solution to the amplified samples (e.g., 2.5 µL of 50% glycerol and 0.2% bromophenol blue; Sambrook et al. 1989). Mix well and load approximately half of the volume of the reaction on a horizontal agarose gel cast in 1¥ TBE buffer. Place DNA size standards (e.g., a mixture of bacteriophage l and fX174 digests) alongside RAPD reactions.
5. Electrophoresis and analysis. Electrophorese gels under standard conditions, typically 5-10 V/cm gel length. After the bromophenol blue dye has reached three fourths of the gel length, the electrophoresis should be complete. Stain gels with ethidium bromide (if not included already in gel and buffer) or use other dyes (SYBR-green; Molecular Probes). Examine and photograph gels under UV light. Depending on the objective of the experiment make a note of polymorphisms, segregating bands, and appearance of overall patterns within fingerprint databases. Return
Comments
Setting up the amplification reactions
When amplifying several DNA samples with a large number of primers, add primers into test tubes first, followed by a master mix containing common reaction components that can include water, buffer, magnesium, dNTPs, polymerase and DNA. Alternatively, if many DNA samples are amplified using one or a few primers, DNA samples are first added to the test tubes on ice followed by the master mix containing the common primer. The tubes are capped and placed in the thermocycler and the cycling is started immediately.
Optimization
RAPD analysis should be carefully optimized. Amplification depends on a number of parameters including magnesium, primer and DNA concentration, thermal cycling conditions (especially annealing temperature), and type and concentration of thermostable polymerase.
RAPD amplification is no longer reproducible below a certain concentration of genomic DNA and produces "smears" or results in poor resolution if DNA concentration is high (Williams et al. 1993). Generally, use 5-25 ng of DNA per 25 µL reaction. However, it is advisable to amplify a dilution series of the template using one or two primers.
DNA polymerases have different requirements for magnesium ions. Therefore, the concentration of magnesium the amplification reaction should be optimized. Consider that the presence of EDTA in TE buffer (up to 1 mM), sometimes used to dissolve genomic DNA, can complex magnesium and reduces its effective concentration. In this case, the magnesium concentration in the reaction should be increased to compensate for this effect. Low magnesium concentrations produce few RAPD bands. The optimal concentration for a particular application should be determined empirically but is ussually within the 1-4 mM range.
RAPD is particularly sensitive to thermal cycling parameters, and therefore depend strongly on the thermocylcer used. Different thermocyclers exhibit different rates of heating and cooling and can show temperature inhomogeneities, even when programmed using identical settings. This may even occur within a same model of thermocycler. Furthermore, thermocyclers that use Peltier devices for heating and cooling can exhibit a decay in the ability to heat and dissipate heat as these unit age. In some units that lack internal temperature probes there can be a considerable difference in temperature between block and sample, and such differences can be important for the success of the RAPD amplification. If possible, it is advisable to use a thermocouple combined with a chart recorder to confirm temperature variation in the thermocycler unit. This should be done at least on an yearly time schedule.
Choice of DNA polymerase
The Taq polymerase holoenzyme was the first enzyme used in RAPD (Williams et al. 1990). However, the Stoffel fragment enzyme (Lawler et al. 1993) has been shown superior for DAF (Bassam et al. 1992) and AP-PCR (Sobral and Honeycutt 1993) analysis. The enzyme provides better reproducibility bu decreases the size distribution of amplification products. The use of this truncated DNA polymerase has also proven superior in RAPD analysis. Return
Applications
RAPD analysis has been successfully used in mapping and fingerprinting applications. Because of considerable concerns on reproducibility of RAPD profiles (Penner et al. 1993, Skroch and Nienhuis 1995), always follow appropriate guidelines for optimization and confirm reproducibility. In genetic mapping, only the RAPD amplification products in coupling with the segregating trait are informative, i.e. amplification products from the parent that carries the dominant form of the gene being mapped (Williams et al. 1993). In these applications, always apply a suitable statistical test to confirm that segregation ratios conform to the expectation (e.g., 1:1 or 3:1 in a backcross or F2 population, respectively). In this and other applications, errors can result from incorrect allele assignment due to small differences in band mobility. It is advisable to use Southern blotting to verify that homologous bands are in fact allelic (Williams et al. 1993).
In fingerprinting applications, the RAPD assay is not an appropriate technique when the difference (localized or dispersed) between the two genomes being compared is limited to an extremely small genomic fraction (generally less than 0.1%). For this purpose use more powerful techniques such as DAF, ASAP or ASAP analysis. However, RAPD can efficiently identify differences that constitute a significant fraction of a genome (e.g., in near-sogenic lines containing 1-10% donor genome segments). Conversely, RAPD markers tend to underestimate genetic distances between distantly related individuals, for example in inter-specific comparisons. Therefore, be cautious when using RAPD for taxonomic studies above the species level. Return
